Closing in on maximum yield of chlorophyll fluorescence using a single multiphase flash of sub-saturating intensity.

Estimation of the maximum chlorophyll fluorescence yield under illumination, or Fm', by traditional single saturation pulse (SP) methodology is prone to underestimation error because of rapid turnover within photosystem (PS) II. However, measurements of fluorescence yield during several single pulses of variable intensity describes the irradiance dependence of apparent Fm', from which estimates of Fm' at infinite irradiance can be derived. While such estimates have been shown to result in valid approximations of Fm', the need to apply several single pulses limits its applicability. We introduce a novel approach that determines the relationship between apparent Fm' and variable irradiance within a single ∼1 s multiphase flash (MPF). Through experiments and simulations, we demonstrate that the rate of variation in irradiance during an MPF is critical for achieving quasi-steady-state changes in the proportions of PSII acceptor side redox intermediates and the corresponding fluorescence yields, which are prerequisites for accurately estimating Fm' at infinite irradiance. The MPF methodology is discussed in the context of improving the accuracy of various parameters derived from chlorophyll fluorescence measurements, such as photochemical and non-photochemical quenchings and efficiencies. The importance of using MPF methodology for interpreting chlorophyll fluorescence, in particular for integrating fluorescence and gas exchange measurements, is emphasized.

Continuous, on-line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus.

A new apparatus for continuously detecting fluorescently labeled DNA fragments is based on infrared fluorescence technology. This technology combines state-of-the-art developments in chemistry, laser technology, and detection, while achieving improved reliability, sensitivity, and flexibility for applications including DNA sequencing. DNA molecules labeled with a novel infrared fluorophore are detected during electrophoresis using a scanning infrared fluorescence microscope. The microscope consists of a laser diode for exciting the fluorophore and a silicon avalanche photodiode for detecting the infrared emission. Optimum conditions for detection and throughput are obtained by adjusting electrophoresis, scanning and imaging parameters. Typical DNA sequencing runs (test templates) allow identification of over 500 bases per sample with greater than 99% accuracy.